Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser¹⁶ or Ser⁶³, and doubly phosphorylated at Ser²⁵ and Ser³⁸, on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser¹⁶ or Ser⁶³ strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser¹⁶ and Ser⁶³ and support the hypothesis that selective targeting by Ser¹⁶-specific or Ser⁶³-specific kinases provides complimentary mechanisms for regulating microtubule function.Stathmin is an 18-kDa ubiquitously expressed microtubule-destabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³ (1–7). Several classes of kinases have been identified that phosphorylate stathmin, including kinases associated with cell growth and differentiation such as members of the mitogen-activated protein kinase (MAPK)2 family, cAMP-dependent protein kinase (1–5, 8–11), and kinases associated with cell cycle regulation such as cyclin-dependent kinase 1 (3, 12–14). Phosphorylation of stathmin is required for cell cycle progression through mitosis and for proper assembly/function of the mitotic spindle (3, 13–16). Inhibition of stathmin phosphorylation produces strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (3, 13–14).Stathmin is known to destabilize microtubules in two ways. One is by binding to soluble tubulin and forming a stable complex that cannot polymerize into microtubules, consisting of one molecule of stathmin and two molecules of tubulin (T2S complex) (17–24). Addition of stathmin to microtubules in equilibrium with soluble tubulin results in sequestration of the tubulin and a reduction in the level of microtubule polymer (17–18, 22, 25–28). In addition to reducing the amount of assembled polymer, tubulin sequestration by stathmin has been shown to increase the switching frequency at microtubule plus ends from growth to shortening (called the catastrophe frequency) as the microtubules relax to a new steady state (17, 29). The second way is by binding directly to microtubules (27–30). The direct binding of stathmin to microtubules increases the catastrophe frequency at both ends of the microtubules and considerably more strongly at minus ends than at plus ends (27). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). These results have led to the suggestion that stathmin might be an important cellular regulator of minus-end microtubule dynamics (27).Phosphorylation of stathmin diminishes its ability to regulate microtubule polymerization (3, 14, 25–26). Phosphorylation of Ser¹⁶ or Ser⁶³ appears to be more critical than phosphorylation of Ser²⁵ and Ser³⁸ for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3, 25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3, 14) suggesting that not only soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could also be regulated in a phosphorylation-dependent manner.It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, specifically, its ability to increase the catastrophe frequency at plus and minus ends due to its direct interaction with microtubules. Thus, we determined the effects of stathmin individually phosphorylated at either Ser¹⁶ or Ser⁶³ and doubly phosphorylated at both Ser²⁵ and Ser³⁸ on dynamic instability at plus and minus ends in vitro at microtubule polymer steady state and physiological pH (pH 7.2). We find that phosphorylation of Ser¹⁶ strongly reduces the direct catastrophe-promoting activity of stathmin at plus ends and abolishes it at minus ends, whereas phosphorylation of Ser⁶³ abolishes the activity at both ends. The effects of phosphorylation of individual serines correlated well with stathmin''s reduced abilities to form stable T2S complexes, to inhibit microtubule polymerization, and to bind to microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not alter the ability of stathmin to modulate dynamic instability at the microtubule ends, its ability to form a stable T2S complex, or its ability to bind to microtubules. The data further support the hypotheses that phosphorylation of stathmin on either Ser¹⁶ or Ser⁶³ plays a critical role in regulating microtubule polymerization and dynamics in cells.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag SDS-PAGE

Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, spine formation, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes down-regulation of Cdk5, whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser⁸ and Thr¹³⁸ as major phosphorylation sites using metabolic labeling of cultured cells followed by two-dimensional phosphopeptide mapping and phosphospecific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the phosphorylation states of p35 in neuronal culture and brain. Using Phos-tag acrylamide, the electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We found a novel phosphorylation site at Ser⁹¹, which was phosphorylated by Ca²⁺-calmodulin-dependent protein kinase II in vitro. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states and quantified Ser⁸, Ser⁹¹, and Thr¹³⁸ phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser⁸ was phosphorylated in all p35 molecules, whereas Ser⁹¹ was phosphorylated in 60% and Thr¹³⁸ was phosphorylated in ∼12% of p35 molecules. These are the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.Phosphorylation is a major post-translational modification of proteins, modulating a variety of cellular functions (1, 2). Because most phosphorylation occurs in a highly site-specific manner, identification of phosphorylation sites has been a subject of intense investigation. Several analytical methods have been utilized to identify phosphorylation sites, including mass spectrometry, amino acid sequencing, and radioisotope phosphate labeling of proteins with mutation(s) at putative phosphorylation site(s) (3, 4). Phosphorylation site-specific antibodies are frequently used to detect phosphorylation at target sites (5, 6). Many phosphospecific antibodies are now commercially available. These phosphospecific antibodies are convenient and useful tools for examining site-specific phosphorylation both in vivo and in vitro. However, they are not appropriate for estimating quantitative ratios of phosphorylation states. Electrophoretic mobility shift on SDS-PAGE is also often used to observe phosphorylation (7–10), but this method is not always applied to site-specific phosphorylation.Phos-tag is a newly developed dinuclear metal complex that can be used to provide phosphate-binding sites when conjugated to analytical materials such as acrylamide and biotin (11). In SDS-PAGE using Phos-tag acrylamide, phosphorylated proteins are trapped by the Phos-tag sites, delaying their migration and thus separating them from unphosphorylated proteins. Subsequent immunoblot analysis with phosphorylation-independent antibodies reveals both the phosphorylated and unphosphorylated bands. Because the migration of the phosphorylated proteins is greatly delayed compared with migration in Laemmli SDS-PAGE, it is easy to identify the phosphorylated proteins from observed positions on blots. In the past 3 years, this method has been used to detect phosphorylation states for many proteins such as ERK1/2, cdc37, myosin light chain, eIF2α, protein kinase D, β-casein, SIRT7, and dysbindin-1 (12–21).Cyclin-dependent kinase 5 (Cdk5)¹ is a proline-directed serine/threonine kinase that is expressed predominantly in postmitotic neurons and regulates various neuronal events such as neuronal migration, spine formation, synaptic activity, and cell death (22–24). Cdk5 is activated by binding to activation subunit p35 and inactivated by proteasomal degradation of p35 (25). In addition, Cdk5 activity is deregulated by cleavage of p35 to p25 with calpain, resulting in abnormal activation and ultimately causing neuronal cell death (26–29). Proteolysis of p35, either by proteasomal degradation or cleavage by calpain, is regulated by phosphorylation of p35 by Cdk5 (30–33). Therefore, phosphorylation of p35 is essential for proper regulation of Cdk5 activity and function. We previously identified Ser⁸ and Thr¹³⁸ as major p35 phosphorylation sites (33). We also showed that phosphorylation of p35 decreased during brain development and proposed its relationship to age-dependent vulnerability of neurons to stress stimuli (32). Thus, to understand the in vivo regulation of Cdk5 activity, it is critical to analyze the phosphorylation states of p35 in brain. However, there is no convenient method to analyze the precise in vivo phosphorylation status of the endogenous proteins.In this study, we applied the Phos-tag SDS-PAGE method to analyze the phosphorylation states of p35 in vivo and in cultured neurons. We constructed standard band profiles of phosphorylated p35 by Phos-tag SDS-PAGE using Ala mutants at Ser⁸ and/or Thr¹³⁸. From these experiments, we observed an unidentified in vivo phosphorylation site at Ser⁹¹. We quantified the phosphorylation at each site in cultured neurons and brain, providing the first quantitative estimate of the in vivo phosphorylation states of p35. We discuss the usefulness of Phos-tag SDS-PAGE to analyze the in vivo phosphorylation states of proteins.     

B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology.Antibodies (Abs)² made by B lymphocytes play a critical role in host defense against infection. Antigen-induced signaling by the B cell receptor (BCR) initiates an activation program that leads to B cell proliferation and subsequent differentiation into Ab-producing cells. BCR clustering by antigens or by anti-immunoglobulin (anti-Ig) Abs used as surrogate antigens initiates multiple signaling pathways that control gene expression, cell survival, and proliferation pathways (1–3).BCR signaling also promotes integrin activation (4, 5), localized actin polymerization, reorganization of the actin cytoskeleton, and changes in B cell morphology (6, 7), all of which may facilitate B cell activation. Integrin activation and cell spreading is critical for the activation of B cells by membrane-bound antigens. Macrophages, dendritic cells, and follicular dendritic cells can present arrays of captured antigens to B cells (8, 9), and this may be one of the main ways in which B cells encounter antigens (10). BCR-induced integrin activation prolongs the interaction between the B cell and the antigen-presenting cell and also allows the B cell to spread on the surface of the antigen-presenting cell such that more BCRs can encounter and bind membrane-bound antigens (11). Subsequent contraction of the B cell membrane allows the B cells to gather the BCR-bound antigen into an immune synapse in which clustered antigen-engaged BCRs are surrounded by a ring of ligand-bound integrins. Formation of this immune synapse reduces the amount of antigen that is required for B cell activation (12, 13).Recent work has shown that B cells in lymphoid organs may contact soluble antigens by extending membrane processes into a highly organized network of lymph-filled conduits (14). These conduits are created by fibroblastic reticular cells that partially ensheathe collagen fibrils. In addition to being rich in collagen, fibronectin, and other extracellular matrix (ECM) components, the fibroblastic reticular cells that form these conduits express high levels of intercellular adhesion molecule-1, the ligand for the α_Lβ₂ integrin (lymphocyte function-associated antigen-1 (LFA-1)) on B cells (10). Thus B cells interacting with these conduits are likely to be in contact with integrin ligands, and integrin-dependent spreading may enhance the ability of B cells to extend membrane processes into the fibroblastic reticular cell conduit.In addition to promoting cell spreading, integrins can act as co-stimulatory receptors that enhance signaling by many receptors including the T cell receptor and the BCR (15–17). Thus signaling proteins that regulate B cell spreading and that are also targets of BCR/integrin co-stimulation may play a key role in the activation of B cells by membrane-bound antigens as well as soluble antigens that are delivered to lymphoid organs by fibroblastic reticular cell conduits.Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related non-receptor protein-tyrosine kinases that integrate signals from multiple receptors and play an important role in regulating cell adhesion, cell morphology, and cell migration in many cell types (18–20). Integrins, receptor tyrosine kinases, antigen receptors, and G protein-coupled chemokine receptors all stimulate tyrosine phosphorylation of Pyk2 and FAK, a modification that increases the enzymatic activity of these kinases and allows them to bind SH2 domain-containing signaling proteins (21). FAK, which is expressed in almost all tissues (21), is a focal adhesion component that mediates integrin-dependent cell migration (22), cell spreading, and cell adhesion (18) in adherent cells as well as co-clustering of LFA-1 with the T cell receptor in lymphocytes (23). Pyk2 is expressed mainly in hematopoietic cells, osteoclasts, and the central nervous system (24) and is critical for chemokine-induced migration of B cells, macrophages, and natural killer cells (20, 25, 26) as well as the spreading of osteoclasts on vitronectin (27). FAK and Pyk2 are thought to mediate overlapping but distinct functions because Pyk2 expression only partially reverses the cell adhesion and migration defects in FAK-deficient fibroblasts (28).In B cells, clustering of the BCR, β₁ integrins, or β₇ integrins induces tyrosine phosphorylation of both Pyk2 and FAK (29–33). FAK is involved in the chemokine-induced adhesion of B cell progenitors (34), and Pyk2 is required for chemokine-induced migration of mature B cells (25). However, the role of these kinases in BCR- and integrin-induced B cell spreading has not been investigated, and the signaling pathways that link the BCR and integrins to tyrosine phosphorylation of Pyk2 and FAK have not been elucidated.We have shown previously that the ability of the BCR to induce integrin activation, B cell spreading, and immune synapse formation requires activation of the Rap GTPases (6, 17). In addition to binding effector proteins such as RapL and Rap1-interacting adaptor molecule (RIAM) that promote integrin activation (35–37), the active GTP-bound forms of Rap1 and Rap2 bind multiple proteins that control actin dynamics and cell morphology (38). Moreover we showed that BCR/integrin-induced phosphorylation of Pyk2 in B cells is dependent on Rap activation (17). However, this previous study did not address how Rap-GTP links the BCR and integrins to Pyk2 phosphorylation, whether Rap activation is important for FAK phosphorylation in B cells, or whether B cell spreading is regulated by Pyk2 or FAK. We now show that Pyk2 and FAK are differentially expressed and localized in B cells, that Pyk2 and FAK are important for B cell spreading, and that integrin engagement enhances BCR-induced phosphorylation of Pyk2 and FAK, a process that depends on both Rap activation and actin dynamics.     

O-Linked N-Acetylglucosamine Modification of Insulin Receptor Substrate-1 Occurs in Close Proximity to Multiple SH2 Domain Binding Motifs

Insulin receptor substrate-1 (IRS-1) is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Ser/Thr kinases impact the metabolic and mitogenic effects elicited by insulin and IGF-1 through feedback and feed forward regulation at the level of IRS-1. Ser/Thr residues of IRS-1 are also O-GlcNAc-modified, which may influence the phosphorylation status of the protein. To facilitate the understanding of the functional effects of O-GlcNAc modification on IRS-1-mediated signaling, we identified the sites of O-GlcNAc modification of rat and human IRS-1. Tandem mass spectrometric analysis of IRS-1, exogenously expressed in HEK293 cells, revealed that the C terminus, which is rich in docking sites for SH2 domain-containing proteins, was O-GlcNAc-modified at multiple residues. Rat IRS-1 was O-GlcNAc-modified at Ser⁹¹⁴, Ser¹⁰⁰⁹, Ser¹⁰³⁶, and Ser¹⁰⁴¹. Human IRS-1 was O-GlcNAc-modified at Ser⁹⁸⁴ or Ser⁹⁸⁵, at Ser¹⁰¹¹, and possibly at multiple sites within residues 1025–1045. O-GlcNAc modification at a conserved residue in rat (Ser¹⁰⁰⁹) and human (Ser¹⁰¹¹) IRS-1 is adjacent to a putative binding motif for the N-terminal SH2 domains of p85α and p85β regulatory subunits of phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2 (PTPN11). Immunoblot analysis using an antibody generated against human IRS-1 Ser¹⁰¹¹ GlcNAc further confirmed the site of attachment and the identity of the +203.2-Da mass shift as β-N-acetylglucosamine. The accumulation of IRS-1 Ser¹⁰¹¹ GlcNAc in HEPG2 liver cells and MC3T3-E1 preosteoblasts upon inhibition of O-GlcNAcase indicates that O-GlcNAcylation of endogenously expressed IRS-1 is a dynamic process that occurs at normal glucose concentrations (5 mm). O-GlcNAc modification did not occur at any known or newly identified Ser/Thr phosphorylation sites and in most cases occurred simultaneously with phosphorylation of nearby residues. These findings suggest that O-GlcNAc modification represents an additional layer of posttranslational regulation that may impact the specificity of effects elicited by insulin and IGF-1.Insulin receptor substrate-1 (IRS-1)1 is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Many of the metabolic and mitogenic effects elicited by insulin and IGF-1 are mediated and modulated by posttranslational modifications of IRS-1, and tight regulation at the posttranslational level is crucial for maintaining insulin sensitivity and controlling growth factor-induced proliferation. Following hormonal stimulation, IRS-1 is phosphorylated by the receptor tyrosine kinases creating SH2 domain docking sites for downstream binding partners including the p85 regulatory subunits of phosphatidylinositol 3-kinase, Grb2, and the tyrosine phosphatase SHP2 (PTPN11) (1). Binding of p85 phosphatidylinositol 3-kinase and Grb2 activate the PI3K/Akt and Ras-MAPK pathways, respectively, whereas binding of SHP2 results in tyrosine dephosphorylation and signal attenuation (2). Positive and negative feedback regulation by Ser/Thr kinases, such as Akt (3), c-Jun N-terminal kinase (JNK) (4), S6K (5), and ERK (6), impact the interactions of IRS-1 with SH2 domain proteins and the receptor thereby affecting the duration and outcome of the signal. IRS-1 has been described as a central node for the integration of information regarding the nutrient and stress status of the cell (7). This information is encoded by site-specific phosphorylation by a number of kinases that regulate the specificity of effects that are elicited following receptor stimulation. Many sites of Ser/Thr phosphorylation have been identified on IRS-1, and cross-talk among Tyr and Ser/Thr phosphorylations at specific residues is evidence of dynamic and complex posttranslational regulation (8, 9). Inappropriate phosphorylation of IRS-1 resulting in the disruption of interactions of IRS-1 with binding partners is implicated in the development of insulin resistance (10) and altered IGF-1 signaling in breast cancer tissue (11, 12).In addition to phosphorylation, Ser/Thr residues in IRS-1 are also dynamically modified by GlcNAc in a nutrient-responsive manner. As opposed to a negatively charged phosphate group, O-GlcNAcylation imparts a bulky, hydrophilic, electrostatically neutral moiety to Ser/Thr residues. The enzymes responsible for the incorporation and removal of the monosaccharide from proteins, O-GlcNAc-transferase and O-GlcNAcase, respectively, are localized in the cytoplasm and the nucleus of all eukaryotic cells (13, 14). Recent studies suggest that the activity of O-GlcNAc-transferase is regulated by insulin (15) and that localization of O-GlcNAc-transferase to the membrane is driven by direct association with phosphatidylinositide 3-phosphate (16). The abundance of O-GlcNAc modification on many proteins in the insulin signaling pathway increases with sustained high glucose and chronic insulin stimulation, and elevated O-GlcNAc modification of IRS-1 correlates with the development of insulin resistance in multiple cell types including 3T3-L1 adipocytes (17, 18), MIN6 pancreatic beta cells (19), Fao rat hepatoma cells (16), human aortic endothelial cells (20), and skeletal muscle (21). The impact of O-GlcNAcylation on insulin signaling and diabetic complications was reviewed recently (22, 23). The direct effect of O-GlcNAc modification on signaling via IRS-1 is not known because conditions that mimic those in the uncontrolled diabetic patient may also result in phosphorylation of IRS-1 at inhibitory sites (16, 24) and O-GlcNAc modification of other proteins in the insulin signaling pathway, such as the insulin receptor, Akt (18), FoxO (25), AMP-activated protein kinase (26), and β-catenin (17).To elucidate site-specific effects of O-GlcNAc modification on IRS-1-mediated signal transduction, we identified the sites of O-GlcNAc modification of rat and human IRS-1 by tandem mass spectrometry. To facilitate detection of the O-GlcNAc-modified peptides and assign the sites of modification, CID coupled with neutral loss-triggered MS3 and electron transfer dissociation (ETD) (27) tandem spectrometric approaches were used. Fragmentation of O-GlcNAc-modified peptides by ETD did not destroy the labile O-linkage (28) permitting direct detection of these peptides by the database searching algorithm ProteinProspector2 (29). O-GlcNAc modification occurred in close proximity to multiple SH2 domain binding motifs and within a region of IRS-1 shown previously to interact with the insulin and IGF-1 receptors (30).     

Analysis of PTEN Complex Assembly and Identification of Heterogeneous Nuclear Ribonucleoprotein C as a Component of the PTEN-associated Complex

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is well characterized for its role in antagonizing the phosphoinositide 3-kinase pathway. Previous studies using size-exclusion chromatography demonstrated PTEN recruitment into high molecular mass complexes and hypothesized that PTEN phosphorylation status and PDZ binding domain may be required for such complex formation. In this study, we set out to test the structural requirements for PTEN complex assembly and identify the component(s) of the PTEN complex(es). Our results demonstrated that the PTEN catalytic function and PDZ binding domain are not absolutely required for its complex formation. On the other hand, PTEN phosphorylation status has a significant impact on its complex assembly. Our results further demonstrate enrichment of the PTEN complex in nuclear lysates, suggesting a mechanism through which PTEN phosphorylation may regulate its complex assembly. These results prompted further characterization of other protein components within the PTEN complex(es). Using size-exclusion chromatography and two-dimensional difference gel electrophoresis followed by mass spectrometry analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a novel protein recruited to higher molecular mass fractions in the presence of PTEN. Further analysis indicates that endogenous hnRNP C and PTEN interact and co-localize within the nucleus, suggesting a potential role for PTEN, alongside hnRNP C, in RNA regulation.Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)⁴ was cloned in 1997 (1–3) and has been well characterized for its tumor-suppressive role by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol 4,5-bisphosphate and antagonizing the phosphoinositide 3-kinase pathway (4–7). PTEN also regulates cell migration, cell cycle progression, DNA damage response, and chromosome stability independently of its lipid phosphatase activity through its potential protein phosphatase activity and/or protein-protein interaction (8–11) (for recent reviews, see 12–14).PTEN is composed of an N-terminal catalytic domain and a C-terminal regulatory domain. The catalytic domain contains a conserved signature motif (HCXXGXXR) found in dual-specific protein phosphatases, and mutations within this catalytic domain, including the C124S mutation, are known to abrogate PTEN catalytic activity (4). The C terminus of PTEN contains a PDZ (PDS-95/Disc-large/Zo-1) binding domain, which interacts with PDZ-containing proteins such as MAGI-1b, MAGI-2, MAGI-3, hDLG, hMAST and NHERF (15–19). In addition to the PDZ binding domain, several key serine and threonine phosphorylation sites (Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵) at the PTEN C terminus are reported to play an important role in regulating its stability, localization, and activity (20–26).Recent studies suggest that PTEN may function within higher molecular mass complexes. Through size-exclusion chromatography, Vazquez et al. (27) demonstrated that PTEN can be separated into two populations: a monomeric hyperphosphorylated subpopulation and a higher molecular mass hypophosphorylated subpopulation. It was hypothesized that PTEN in its dephosphorylated form can interact with PDZ-containing proteins such as hDLG and be recruited into a higher molecular mass complex. Although the components within PTEN complex(es) have not been systematically studied and purified, MAGI-2, hDLG (27), NHERF2, PDGFR (19), NEP (28), and MVP (29) have been identified as potential components of the PTEN complex using the same size-exclusion chromatography methodology.In this paper, we aim to (i) investigate the essential elements of PTEN required for its complex formation and (ii) dissect the components of the PTEN-associated complex(es). Our results indicate that PTEN catalytic activity or its PDZ binding domain is not absolutely required for complex assembly. PTEN phosphorylation status on amino acids Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵, on the other hand, has a significant role in complex formation. In addition, we demonstrate that the PTEN complex is enriched in nuclear lysates, which suggests a mechanism through which phosphorylation can regulate complex assembly. Using two-dimensional difference gel electrophoresis (DIGE) analysis and comparing proteins present in higher molecular mass fractions in the presence and absence of PTEN followed by mass spectrometry analysis, we have identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a potential component within the PTEN complex. PTEN and hnRNP C are shown here to interact and co-localize in the nucleus. We hypothesize that the PTEN and hnRNP C complex may play a role in RNA regulation.     

Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A₂ mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser¹¹⁷⁷ and Akt at Ser⁴⁷³. These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser³⁸⁰/Thr^382/383 PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G_i family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser³⁸⁰/Thr^382/383 phosphorylation, and dephosphorylation of both Akt (at Ser⁴⁷³) and eNOS (at Ser¹¹⁷⁷). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.Insulin exerts multiple biological actions relating to not only metabolism but also to endothelial functions (1, 2). Insulin has beneficial effects on the vasculature, primarily because of its ability to enhance endothelial nitric-oxide synthase (eNOS)² activation and expression. These effects, in turn, enhance the bioavailability of nitric oxide (3), which engenders a wide array of antiatherogenic effects. Global insulin resistance is a key feature of the metabolic syndrome leading to cardiovascular disease. In an insulin-resistant state, a systemic deregulation of the insulin signal leads to a combined deregulation of insulin-regulated metabolism and endothelial functions (4), resulting in glucose intolerance and cardiovascular disease. Insulin resistance is associated with endothelial dysfunction (5), a hallmark of atherosclerosis, and predicts adverse cardiovascular events (6). Therefore, endothelium-specific insulin resistance (impaired insulin-stimulated phosphorylation of Akt and eNOS) may play an important role in the development of cardiovascular diseases.Prostanoids have critical roles in the development of endothelial dysfunction (7). Thromboxane A₂ (TXA₂) is believed to be a prime mediator of a variety of cardiovascular and pulmonary diseases such as atherosclerosis, myocardial infarction, and primary pulmonary hypertension. TXA₂ perturbs the normal quiescent phenotype of endothelial cells (ECs). This results in leukocyte adhesion to the vessel wall as well as increased vascular permeability and expression of adhesion molecules on ECs, all important components of the inflammatory response. In smooth muscle cells, TXA₂ promotes proliferation (8) and migration, contributing to neointima formation (9). TXA₂ binds to the thromboxane A₂ receptor (TP), which has two isoforms TPα and TPβ in human (10–12), activation of which is implicated in atherosclerosis and inflammation (13–16). Atherosclerosis is accelerated by diabetes and is associated with increased levels of TXA₂ and other eicosanoids that stimulate TP (14). TP expression and plasma levels of TP ligands are elevated, both locally and systemically, in several vascular and thrombotic diseases (17). Importantly, TP activation induces EC apoptosis (15, 18) and prevents tube formation (19) by inhibiting Akt phosphorylation (18). TP activation also inhibits vascular endothelial growth factor-induced EC migration and angiogenesis by decreasing Akt and eNOS phosphorylation (20). However, the regulatory mechanisms underlying Akt inhibition by TP stimulation remain largely undefined. Moreover, whether TP activation impairs endothelial insulin signaling is also unclear.Here, we investigated whether TP ligands interfere with insulin signaling. Our results reveal that activation of TP using a potent and stable ligand (IBOP) abrogates insulin signaling in ECs. We also show that Rho/ROCK/LKB1-mediated PTEN (phosphatase and tensin homolog deleted on chromosome ten) up-regulation is required for TP-induced inhibition of insulin signaling in ECs.     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

Epidermal Growth Factor Receptor Phosphorylation Sites Ser991 and Tyr998 Are Implicated in the Regulation of Receptor Endocytosis and Phosphorylations at Ser1039 and Thr1041

Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser⁹⁹¹ and Tyr⁹⁹⁸ accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser¹⁰³⁹ and Thr¹⁰⁴¹. These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195–4206). EGF-induced phosphorylations at Ser¹⁰³⁹ and Thr¹⁰⁴¹ were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr⁹⁹⁸, Ser⁹⁹¹, Ser¹⁰³⁹, and Thr¹⁰⁴¹ governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.Upon activation by ligand, the epidermal growth factor receptor (EGFR)¹ dimerizes, sometimes as heterodimers with other EGFR family members; is catalytically activated by reorientation of kinase region subdomains; becomes covalently modified by phosphorylation and ubiquitination; and interacts with a variety of intracellular proteins (1, 2). These events activate intracellular signaling cascades, and concurrently the dimerized receptors become internalized through endocytosis and then may be recycled to the cell surface or degraded in lysosomes (3). Systematic analysis of EGFR family phosphorylation-dependent protein interactions has been assessed (4, 5), and many of the known EGFR-interacting proteins can be categorized as functioning in cellular processes such as EGF-induced signal transduction and EGFR endocytosis and trafficking. Temporal analysis of tyrosine phosphorylation following EGF treatment of cells revealed groups of EGFR substrates with shared profiles of phosphorylation kinetics, including some that display rapid kinetics of phosphorylation accumulation and are involved in signal transduction (e.g. ERK kinase activation) and others that accumulate more slowly following ligand treatment and are involved in receptor internalization and down-regulation (5–11). Although advances in MS and the definition of phosphorylation-dependent protein-protein interactions have led to a greatly expanded view of EGFR function and regulation, our understanding of the biological consequences and spatial-temporal relationships of individual modifications is incomplete.In a previous quantitative phosphoproteomics study aimed at the identification of drug-modulated changes in phosphorylation associated with the EGFR network, a cluster of three sites of phosphorylation in the EGFR carboxyl tail region was identified as affected by receptor stimulation by EGF and inhibited by the ATP-competitive EGFR inhibitor PKI166 in human A431 tumor cells and xenograft tumors (12). The three sites in the cluster, Ser⁹⁹¹,² Ser⁹⁹⁵, and Tyr⁹⁹⁸, are localized within a single tryptic peptide having the sequence MHLPSPTDSNFYR that spans residues 987–999. The phosphorylation of Tyr⁹⁹⁸ was first described by Stover et al. (12), whereas the two serine sites were shown previously to be phosphorylated by Heisermann and Gill (13). Numerous recent studies using different cultured cell models have verified the phosphorylation of EGFR at Tyr⁹⁹⁸ and Ser⁹⁹¹ (10, 11, 14, 15), and Thr⁹⁹³ was also observed to be phosphorylated within this same region of the EGFR in EGF-stimulated HeLa cells (10). The modulation of these sites by EGF and the EGFR inhibitor implicates them in EGFR signaling and suggests that they may have utility as pharmacodynamic markers of EGFR activity. However, the function and importance of these sites, their modulation by kinases and phosphatases, and possible roles in EGFR function remain unknown.Several amino acid residues in the EGFR have been implicated in the regulation of its trafficking. Sorkin et al. (16) showed that substitution of Tyr⁹⁹⁸ with phenylalanine rendered high density EGFRs defective for endocytosis and interaction with AP-2. More recent kinetic studies using MS indicated that EGFR phosphorylation at both Tyr⁹⁹⁸ (5) and Ser⁹⁹¹ (10) occurs relatively slowly compared with other EGF-induced tyrosine phosphorylations known to be involved in receptor-proximal signal transduction. For example, Mann and co-workers (10) recorded maximal phosphorylation at EGFR sites Tyr¹¹¹⁰, Tyr¹¹⁷², and Tyr¹¹⁹⁷ at 1 min post-EGF, whereas EGF-stimulated phosphorylation at Tyr⁹⁹⁸ was still increasing at 15 min post-EGF (5), and a peptide containing both Ser(P)⁹⁹¹ and Thr(P)⁹⁹³ peaked after 10 min (10). However, the role of phosphorylation at Tyr⁹⁹⁸ and Ser⁹⁹¹ has not been reported. Another region of the EGFR, spanning residues 1026–1046, was identified by Zwang and Yarden (17) as a target of phosphorylation downstream of the stress-activated mitogen-activated protein (MAP) kinase p38 and associated with transient internalization and recycling of the EGFR in response to cytokine (TNFα) and stress challenges such as UV irradiation and the chemotherapeutic agent cisplatinum. Within this part of the receptor, a 13-residue section spanning 1029–1041 and the leucines at 1034 and 1035 in particular were found to be essential for ligand- and dimerization-induced EGFR endocytosis (18). Although both EGF- and stress-induced EGFR internalization may be clathrin-mediated, they differ in that the former leads to receptor down-regulation and involves the E3 ubiquitin ligase CBL (19), whereas the latter involves receptor recycling, is not associated with receptor phosphorylation at the CBL binding site Tyr(P)¹⁰⁶⁹, and, in the case of TNFα treatment, involves activation of the transforming growth factor β-activated kinase TAK1 upstream of p38 (20). Interestingly although p38 kinase is not required for EGF-induced EGFR internalization, it is required for CBL-dependent receptor degradation (21). Therefore, alternate pathways involving p38 kinase regulate the down-regulation or recycling of the EGFR in response to diverse extracellular signals. However, the molecular details that govern these two processes are not fully understood.In the current study, EGFR phosphorylation, signaling, protein-protein interactions, and trafficking were analyzed to address the role of Tyr⁹⁹⁸ and Ser⁹⁹¹ in EGFR endocytosis. This was achieved by application of complementary methods including quantitative selected reaction monitoring (SRM, also known as MRM for multiple reaction monitoring) mass spectrometry, fluorescence imaging and cell sorting, immunoaffinity protein enrichment and blotting, and site-directed mutagenesis. Substitution mutations that prevented phosphorylation at EGFR Tyr⁹⁹⁸ and Ser⁹⁹¹ did not prevent EGFR-to-ERK signaling but impaired EGF-induced receptor internalization and stimulated p38 kinase-dependent receptor phosphorylation at positions Ser¹⁰³⁹ and Thr¹⁰⁴¹. These findings confirm the importance of Tyr⁹⁹⁸ and reveal a role for Ser⁹⁹¹ in EGF-mediated EGFR internalization possibly involving cross-talk with the p38 kinase-dependent EGFR recycling pathway.     

Dual Regulation of Muscle Glycogen Synthase during Exercise by Activation and Compartmentalization

Glycogen synthase (GS) is considered the rate-limiting enzyme in glycogenesis but still today there is a lack of understanding on its regulation. We have previously shown phosphorylation-dependent GS intracellular redistribution at the start of glycogen re-synthesis in rabbit skeletal muscle (Prats, C., Cadefau, J. A., Cussó, R., Qvortrup, K., Nielsen, J. N., Wojtaszewki, J. F., Wojtaszewki, J. F., Hardie, D. G., Stewart, G., Hansen, B. F., and Ploug, T. (2005) J. Biol. Chem. 280, 23165–23172). In the present study we investigate the regulation of human muscle GS activity by glycogen, exercise, and insulin. Using immunocytochemistry we investigate the existence and relevance of GS intracellular compartmentalization during exercise and during glycogen re-synthesis. The results show that GS intrinsic activity is strongly dependent on glycogen levels and that such regulation involves associated dephosphorylation at sites 2+2a, 3a, and 3a + 3b. Furthermore, we report the existence of several glycogen metabolism regulatory mechanisms based on GS intracellular compartmentalization. After exhausting exercise, epinephrine-induced protein kinase A activation leads to GS site 1b phosphorylation targeting the enzyme to intramyofibrillar glycogen particles, which are preferentially used during muscle contraction. On the other hand, when phosphorylated at sites 2+2a, GS is preferentially associated with subsarcolemmal and intermyofibrillar glycogen particles. Finally, we verify the existence in human vastus lateralis muscle of the previously reported mechanism of glycogen metabolism regulation in rabbit tibialis anterior muscle. After overnight low muscle glycogen level and/or in response to exhausting exercise-induced glycogenolysis, GS is associated with spherical structures at the I-band of sarcomeres.The desire to understand metabolism and its regulation dates back several centuries, but it has exponentially increased during the last decades in an effort to treat or prevent type 2 diabetes mellitus (T2DM).3 Defective muscle glycogen synthesis has been repeatedly reported in patients with T2DM (1–3). Several studies have shown impairments of insulin-induced glycogen synthase (GS) activation in skeletal muscle from T2DM patients and in healthy subjects at increased risk for T2DM, such as healthy obese and first-degree relatives of patients with T2DM (4–9).The first scientific studies on GS date from the 1960s, but still today there is a lack of understanding on its regulation. GS is the rate-limiting enzyme in glycogenesis and is classically used as an example of an allosterically and covalently regulated enzyme. It is well accepted that GS is complexly regulated by sequences of hierarchal phosphorylations (10) in at least nine sites and by its allosteric activator, glucose 6-phosphate (G6P) (11, 12). However, the exact effects of GS phosphorylation at different sites on its regulation are still not clear. GS phosphorylation sites are distributed between the NH₂- and the COOH-terminal domains. The NH₂ terminus domain contains two sites, 2 (Ser⁷) and 2a (Ser¹⁰), that are phosphorylated in a hierarchal mode. Phosphorylation of site 2 is needed as a recognition motif for casein kinase 1 to phosphorylate site 2a (13, 14). Several protein kinases have been reported to phosphorylate site 2 in vitro, among them PKA, CaMKII, PKC, AMPK, GPhK, and MAPKAPKII (15–17). At the COOH terminus of muscle GS, there are at least seven phosphorylation sites; sites 3a (Ser⁶⁴⁰), 3b (Ser⁶⁴⁴), 3c (Ser⁶⁴⁸), 4 (Ser⁶⁵²), 5 (Ser⁶⁵⁶), 1a (Ser⁶⁹⁷), and 1b (Ser⁷¹⁰). Sites 3, 4, and 5 are phosphorylated in a hierarchal mode. Casein kinase II phosphorylates site 5, establishing a recognition motif for GSK-3 to phosphorylate sequentially sites 4, 3c, 3b, and 3a (18–21). Dephosphorylation of sites 2 and 3 increase GS intrinsic activity much more than dephosphorylation of the remaining sites, which have little or no effect on the enzyme activity (22). The effect of GS phosphorylation at sites 1a and 1b remains elusive. G6P binding reverses covalent inactivation of GS by phosphorylation (11) and increases susceptibility of the enzyme to be activated by the action of protein phosphatases (23), mainly by glycogen-targeted protein phosphatase 1.Intracellular compartmentalization of GS has been reported in several studies. In isolated hepatocytes, incubation with glucose induces GS activation and intracellular translocation to the cell periphery (24). In contrast, in the absence of glucose, GS has been shown to be mainly located inside the nucleus of both cultured liver and muscle cells; however, following addition of glucose GS translocates to the cytosol (25). In a previous study, we reported a novel regulatory mechanism of skeletal muscle glycogen metabolism (26). We showed that severe glycogen depletion induced by muscle contraction leads to rearrangement of cytoskeleton actin filaments to form dynamic intracellular compartments. Both GS and phosphorylase associate with such compartments to start glycogen re-synthesis. Furthermore, we showed that GS phosphorylated at site 1b (P-GS1b) was located at the cross-striations, the I-band of sarcomeres, whereas when phosphorylated at sites 2+2a (P-GS2+2a), GS formed some clusters homogeneously distributed along muscle fibers. In the present study we investigate the existence and relevance of such regulatory mechanism in human muscle metabolism.     

Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcγ receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcγ receptor-mediated phagocytosis to enhance the innate immune response.Receptors that interact with the constant region of IgG (FcγR)⁴ mediate the recognition and elimination of soluble immune complexes and particles coated (opsonized) with immunoglobulins. Clustering of FcγR on the surface of leukocytes upon attachment to multivalent ligands induces their activation and subsequent internalization. Soluble immune complexes are internalized by endocytosis, a clathrin- and ubiquitylation-dependent process (1). In contrast, large, particulate complexes like IgG-coated pathogens are ingested by phagocytosis, a process that is contingent on extensive actin polymerization that drives the extension of pseudopods (2). In parallel with the internalization of the opsonized targets, cross-linking of phagocytic receptors triggers a variety of other responses that are essential components of the innate immune response. These include degranulation, activation of the respiratory burst, and the synthesis and release of multiple inflammatory agents (3, 4).Like T and B cell receptors, FcγR possesses an immunoreceptor tyrosine-based activation motif that is critical for signal transduction (3, 4). Upon receptor clustering, tyrosyl residues of the immunoreceptor tyrosine-based activation motif are phosphorylated by Src family kinases, thereby generating a docking site for Syk, a tyrosine kinase of the ZAP70 family (3, 4). The recruitment and activation of Syk in turn initiates a cascade of events that include activation of Tec family kinases, Rho- and ARF-family GTPases, phosphatidylinositol 3-kinase, phospholipase Cγ (PLCγ), and a multitude of additional effectors that together remodel the underlying cytoskeleton, culminating in internalization of the bound particle (5, 6).Phosphoinositide metabolism is thought to be critical for FcγR-induced phagocytosis (7, 8). Highly localized and very dynamic phosphoinositide changes have been observed at sites of phagocytosis: phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) undergoes a transient accumulation at the phagocytic cup, which is rapidly superseded by its complete elimination from the nascent phagosome (7). The secondary disappearance of PtdIns(4,5)P₂ is attributable in part to the localized generation of phosphatidylinositol 3,4,5-trisphosphate, which has been reported to accumulate at sites of phagocytosis (9). Activation of PLCγ is also believed to contribute to the acute disappearance of PtdIns(4,5)P₂ in nascent phagosomes. Indeed, the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate has been detected by chemical means during FcγR-evoked particle ingestion (10, 11). Moreover, imaging experiments revealed that DAG appears at the time and at the precise site where PtdIns(4,5)P₂ is consumed (7).Two lines of evidence suggest that the DAG generated upon engagement of phagocytic receptors modulates particle engulfment. First, antagonists of PLC severely impair phagocytosis by macrophages (7, 12). This inhibition is not mimicked by preventing the associated [Ca²⁺] transient, suggesting that DAG, and not inositol 1,4,5-trisphosphate, is the crucial product of the PLC (13). Second, the addition of exogenous DAG or phorbol esters, which mimic the actions of endogenous DAG, augment phagocytosis (14, 15).Selective recognition of DAG by cellular ligands is generally mediated by specific regions of its target proteins, called C1 domains (16). Proteins bearing C1 domains include, most notably, members of the classical and novel families of protein kinase C (PKC), making them suitable candidates to account for the DAG dependence of phagocytosis. Indeed, PKCα, a classical isoform, and PKCϵ and PKCδ, both novel isoforms, are recruited to phagosomes (12, 15, 17, 18). Although the role of the various PKC isoforms in particle engulfment has been equivocal over the years, Cheeseman et al. (12) convincingly demonstrated that PKCϵ contributes to particle uptake in a PLC- and DAG-dependent manner.PKCs are not the sole proteins bearing DAG-binding C1 domains. Similar domains are also found in several other proteins, including members of the RasGRP family, chimaerins, and Munc-13 (19–21). One or more of these could contribute to the complex set of responses elicited by FcγR-induced DAG production. The RasGRP proteins are a class of exchange factors for the Ras/Rap family of GTPases (22). There are four RasGRP proteins (RasGRP1 to -4), and emerging evidence has implicated RasGRP1 and RasGRP3 in T and B cell receptor signaling (23–27).The possible role of DAG-mediated signaling pathways other than PKC in phagocytosis and the subsequent inflammatory response has not been explored. Here, we provide evidence that DAG stimulates Ras and Rap1 at sites of phagocytosis, probably through RasGRPs. Last, the functional consequences of Ras and Rap1 activation were analyzed.